PROJECT SUMMARY/ABSTRACT Skeletal stem cells (SSCs) reside in the bone marrow and periosteum (outer layer of bone) and contribute to the lifelong regeneration of bone and cartilage, making them as a promising therapeutic target for degenerative bone diseases and bone defects. These broadly distributed bone forming stem cells are likely to be heterogeneous, and yet there is no molecular marker that specifically defines their population in vivo. Hence, genetically defining, characterizing, and manipulating SSCs has been a tremendous challenge. The functional differences in different subpopulations of SSCs, as well as specific factors and molecules that regulate different SSC subpopulations in different tissue locations, are essentially unknown. These obstacles have limited our ability to discover better ways to manipulate and improve endogenous SSC functions with the goal of reversing conditions of degenerative bone diseases and aged bone defects. Age is a significant risk factor for many disorders of bone and cartilage, such as osteoporosis and arthritis. Although the clinical changes in bone and cartilage with age have been extensively studied, the underlying causes remain elusive. Like other age-associated functional declines, at least some of the defects in bones and cartilage in the elderly have been attributed to changes in the populations and functions of SSCs. However, due to the challenges described above, the age-associated changes in SSC subpopulation composition, as well as cellular and molecular changes within SSC populations remain poorly understood. The goal of this proposal is to molecularly define the in vivo identity of the SSC population, to characterize SCC subpopulations (SCC heterogeneity), and to examine changes in the SSC population associated with age in mice. Our previous studies showed that periosteal SSCs can be genetically defined by the myxovirus resistance-1 (Mx1) marker and the alpha smooth muscle actin (?SMA) mesenchymal marker. Furthermore, we found that Mx1+?SMA+ periosteal SSCs, rather than Nestin-GFP+ bone marrow SSCs, rapidly respond to injury and provide new osteoblasts for injury repair in vivo. Hence, periosteal SSCs are critical for the lifelong replenishment of injury-repairing osteoblasts in vivo. Using these SSCs as a model and positive control, we plan to achieve the goal of this project through the following aims. First, using the latest single-cell RNA sequencing (RNA-seq) technology, to perform a comprehensive profiling and identification of the stem cell population in mouse periosteal tissue. Cell types, the hierarchal structure of periosteal cells, and molecular characteristics of periosteal SSCs and their subpopulations will be defined by single-cell RNA-seq. Purified Mx1+?SMA+ periosteal SSCs will be used as a reference and positive control. For comparison, we will perform a similar analysis for bone marrow SSCs to further define the molecular differences between these two different populations of SSCs. Second, to determine age-associated changes in SSCs and their subpopulations, we will perform single-cell RNA-seq for periosteal cells isolated from aged (2-year old) mice. Changes in transcriptome between young and old periosteal SSCs will be characterized, including age-associated transcriptomics features like cryptic transcription. Upon completion of this work, we will achieve new biological insights into the regulation of different SSC subsets in different locations, providing new therapeutic targets for reversing bone diseases and defects.